Carol M. Troy - US grants

This project deals with the cell and molecular biology of a newly described neuronal intermediate filament and its relationship to the neurofilament system. The ontogeny of peripherin and the neurofilament triplet proteins will be studies in mouse embryo using immunostaining. The relationship of these two filament systems in development will be examined with double- labeling, allowing construction of 3-D models. Interactions between the neurofilament triplet and peripherin will be investigated in an NF-rich, peripherin-negative system and in an NF-poor, peripherin-rich system, and in mixed systems; using double-labeling immunofluorescence staining and electron microscopy. The function of peripherin in neuronal differentiation will be studied by transfection of PC12 cells with specific DNA sense and anti-sense constructs. The regulation of expression of the mRNAs for peripherin and the neurofilament triplet protein by nerve growth factor will be examined in PC12 cells using 32P- labelled cDNA probes. The results of these studies will give insight into the roles of these two different filament systems in neuronal function and differentiation. The role of peripherin in neurological diseases will be investigated in model systems for neurodegenerative disorders and in human neuropathological specimens of motor neuron disease.

DESCRIPTION (adapted from applicant's abstract) Neuronal cell death is a prominent feature of many diseases, including Alzheimer's disease and amyotrophic lateral sclerosis. The long term goals of this project are to decipher the molecular mechanisms leading to apoptotic neuronal cell death. In particular, the investigators are interested in the mechanisms by which oxidative stress leads to cell death. The specific studies proposed here arise from experiments using cultures of PC12 cells and sympathetic neurons as models to probe the means by which oxidative stress causes neuronal death. An understanding of the mechanisms of these pathways would allow the design of specific therapies. The applicants will contrast and compare two different paradigms of apoptotic cell death: free radical mediated cell death and trophic factor withdrawal mediated cell death in the same neuronal cell types, PC12 cells and sympathetic neurons. By utilizing the same neuronal cells to study these two pathways they eliminate the possibility that differences in the pathways are the result of availability of different death promoting molecules. They have developed the hypotheses that nitric oxide and interleukin-1B are required for free radical mediated death to occur. They will examine these hypotheses with the following specific aims: 1. To determine how NO mediates free radical induced neuronal cell death: a. Which NOS species are necessary for cell death? b. Does NO induce p53 activity. c. Is PARP activated in free radical induced cell death? d. Is peroxynitrite a critical species in NO mediated cell death? 2. To determine how IL-1B mediates cell death induced by the down-regulation of SOD1 while it has no deleterious effects in the trophic factor deprivation paradigm. a. Does IL-1B increase NOS in PC12 cells? b. What are the IL-1 receptor levels on PC12 cells? Are they regulated in cell death? c. Are neurons from IL-1 receptor knockout mice protected from oxidative stress? d. Is IL-1B increased in bcl-2 transfected PC12 cells after V-ASOD1 treatment? These studies will enhance our understanding of how NO and IL-1B operate in apoptotic neuronal cell death. By deciphering the apoptotic death pathway we will have better insight into the treatment of diseases in which there is aberrant cell death.

DESCRIPTION (provided by applicant): Our overall aim is to determine the molecular mechanisms of beta-amyloid-induced neuronal death. Deposition of insoluble Abeta, together with tangle formation and neuronal loss, are hallmarks of Alzheimer's disease. Recent studies show that increased Abeta induces synaptotoxicity, a parameter which correlates with cognitive decline in AD. Evidence from our work and from other laboratories shows that insoluble Abeta induces apoptosis in cultured neurons. The Abeta-mediated death pathway has many similarities to the trophic factor deprivation (TFD) mediated death pathway. These include induction of c-fos and c-jun, use of cell cycle components, activation of the JNK cascade, and protection by bcl-2. We have clearly demonstrated that caspase-2 is necessary for Abeta as well as for TFD mediated death and that although there is parallel activation of caspase-3 it is not sufficient to induce death. However, data obtained from caspase-2 null mice indicate that the death pathways of these two stimuli are not identical: cultured sympathetic neurons from these mice are protected from Abeta but not from TFD. We propose the hypothesis that Abeta and TFD both execute death via a caspase-2 mediated pathway but that the relative expression of caspase regulators (IAPs and Diablo/SMAC) determines different alternative pathways for Abeta and TFD. We additionally propose the hypothesis that JNK activation leads to induction of Fas and recruitment of RAIDD activating caspase-2. By contrasting and comparing the mechanisms employed by these two death stimuli we can further dissect the Abeta-mediated death pathway. We will examine these hypotheses with the following specific aims: 1. To determine which caspases are activated (processed) during Abeta and TFD mediated death and which caspases can execute Abeta and TFD induced death. 2. To determine whether there is differential expression and regulation of MIAP3 or Diablo after Abeta or TFD. 3. To determine whether activation of the JNK cascade leads to caspase-2 activation. 4. To determine whether the molecular components of the Abeta death pathway, identified in the preceding Aims, are altered in a mouse model of AD and/or in human AD brain.

DESCRIPTION (provided by applicant): Neuronal degeneration and death are the hallmarks of many neurological diseases and there is considerable evidence that oxidative stress plays an important role in stroke/ischemia. The long term goals of this project are to elucidate the molecular mechanisms governing free radical-initiated apoptotic neuronal death and to utilize this molecular data for the design of therapeutic interventions. We have used the down-regulation of SOD1 in cultured primary neurons as a model of oxidative stress in which the pathways leading to neuronal apoptosis can be examined. Down-regulation of SOD1 leads to a death via a peroxynitrite-mediated pathway. This pathway requires activation of caspase-1 which in turn leads to the generation of IL-1beta which is released from the cell and acts in an autocrine manner on the IL-1 receptor to potentiate cell death. We also have evidence that activation of caspases-8 and -7, but unexpectedly not caspase-3, are required for death to proceed. This suggests a novel caspase cascade in which caspase-1 is the apical caspase leading to autocrine receptor activation followed by activation of caspase-8 and finally to activation of the executioner caspase, caspase-7. The failure to activate caspase-3 has not previously been seen in neuronal death cascades and supports the feasibility of designing specific agents to intervene in this pathway. We now propose the hypotheses that there is a feedback loop of NO and IL-1beta which is an essential component of the free radical death pathway and that there is a novel caspase cascade initiated by caspase-1 activation. These hypotheses will be examined with the following specific aims: 1. To determine the time course of induction/activation and localization of key molecules in the SOD1 down-regulation pathway and in ischemia. 2. To determine how the induction of nNOS is regulated after SOD1 down-regulation and after ischemia. 3. To determine how the induction of IL-1beta is regulated after SOD1 down-regulation and after ischemia. 4. To determine how caspases-8 and -7 are activated after SOD1 down-regulation. 5. To determine if there is activation/regulation of caspase-3 and other caspases after SOD1 down-regulation.

Our overall aim is to determine the molecular mechanisms of [unreadable]-amyloid (A[unreadable])-induced synaptic loss and neuronal death. Deposition of insoluble A[unreadable], together with tangle formation, loss of synapses and neurons, are hallmarks of Alzheimer[unreadable]s disease. More recently soluble A[unreadable] oligomeric species have been found in AD and these may correlate with synaptic loss. While the debate continues about the role of each of these in the disease it is important to develop model systems where these processes can be studied. Studies show that increased A[unreadable] induces synaptotoxicity, a parameter which correlates with cognitive decline in AD. Evidence from our work and from other laboratories shows that aggregated and oligomeric A[unreadable] induce apoptosis in cultured neurons and that sublethal concentrations of A[unreadable] induce changes in synapse morphology in primary neurons and brain slices. Our work shows that A[unreadable] induces activation of caspase-2 and -3 but that only caspase-2 executes death. Caspase-2 and its downstream target Bim are increased in AD brains. We are proposing that, in neurons exposed to A[unreadable], the main function of caspase-3 is the regulation of synaptic plasticity, not the execution of cell death; caspase-2 executes death. We propose the hypothesis that there is dose-dependent activation of different caspases by A[unreadable] leading to synaptic remodeling, synaptic loss and neuronal death. Sublethal doses of A[unreadable] activate caspase-3; caspase-3 in this setting does not execute death but is responsible for remodeling synapses as a protective mechanism in response to A[unreadable]; the activity of caspase-3 is modulated by IAPs. With increasing time of exposure or increasing levels of A[unreadable], synapse pruning becomes excessive, leading to synaptotoxicity which in turn induces trophic factor deprivation leading to further synaptic loss and eventually to activation of caspase-2 and neuronal death. Lethal doses of A[unreadable] activate caspase-2 and caspase-3; caspase-2 induces Bim and executes the neuron, caspase-3 activity is inhibited from executing death by cIAP1. Different complexes serve to regulate caspase-2 activity in neurons. Caspase-2 activation requires RAIDD; PIDD complexes with RAIDD in healthy neurons to prevent caspase-2 activation. We will examine these hypotheses using primary hippocampal neuron cultures and mouse models of neurodegeneration, with the following specific aims: 1. To determine how caspases regulate synaptic loss induced by A[unreadable]. 2: To determine how caspases are regulated and activated by A[unreadable] and TFD. 3: To determine how caspase-2 regulates the induction of Bim after A[unreadable] treatment.

DESCRIPTION (provided by applicant): Stroke is the 3rd largest cause of death and the largest cause of disability in the U.S., yet there are no effective therapies for the vast majorityof cases. Present therapeutic options merely aim to restore blood flow in the hopes of salvaging at risk tissue, but offer no targeted neuroprotection. Development of neuroprotective therapies has been hindered by lack of knowledge of the signaling pathways critical in secondary injury following ischemic stroke. For more than a decade, the caspase family of death proteases has been implicated in cerebral ischemia and neurodegeneration. Recent evidence shows that distinct caspase pathways are activated during ischemia. We have identified the caspase-9/-6 pathway as responsible for neuronal dysfunction and death after ischemia. Our data show that targeting caspase-9 activity provides substantial neuroprotection following an ischemic insult. Moreover, we find that caspase-9 activity is required for two aspects of ischemic pathogenesis: 1) neuronal degeneration and 2) the development of cerebral edema. Edema is caused by a loss of vascular integrity, rather than death of endothelial cells and pericytes in the blood vessels (BV). The elimination of tight junctions between these cells allows extravasation of fluid from small intracranial BVs. Edema formation is a major contributor to death and disability in severe stroke. Medical therapies and surgical decompressive procedures have only minimally altered the natural history of this pathogenic process. In our studies, a cell permeant caspase-9 inhibitor, Pen1-XBIR3, reduces caspase-9 activity and concomitantly abolishes edema. This finding opens the question of whether caspase-9 activity is a direct cause of edema through the impairment of vascular integrity of small cerebral BVs. Our preliminary data suggest that active caspase-9 regulates edema by decreasing the expression of matrix metalloproteinase 9 (MMP-9). Our data also show that expression of the precursor of mature NGF, proNGF, increases during stroke. ProNGF is a high affinity ligand for p75NTR, and we have shown that signaling through p75NTR activates caspase-9. p75NTR is found in small BVs in the brain, and expression of p75NTR increases during stroke. Our preliminary data also show that activated caspase-9 is present in BVs, and that caspase-9 inhibition prevents the stroke-induced expression of MMP-9. We now propose the hypothesis that the development of edema in stroke is mediated by proneurotrophin (proNT) signaling through p75NTR, which activates caspase-9 in small BVs to cleave substrates vital to the integrity of the vessels. We will utilize in vivo an in vitro models to examine this hypothesis with the following Specific Aims: Aim 1: To determine if induction of proNTs triggers caspase-9 activation in small BVs. Aim 2: To determine if signaling via p75NTR activates caspase-9 and leads to edema. Aim 3: To determine how caspase-9 cleavage of substrates leads to loss of vascular integrity.

DESCRIPTION (provided by applicant): Stroke is the 3rd largest cause of death and the largest cause of disability in the U.S., yet there are no effective therapies for the vast majorityof cases. Present therapeutic options merely aim to restore blood flow in the hopes of salvaging at risk tissue, but offer no targeted neuroprotection. Development of neuroprotective therapies has been hindered by lack of knowledge of the signaling pathways critical in secondary injury following ischemic stroke. For more than a decade, the caspase family of death proteases has been implicated in cerebral ischemia and neurodegeneration. Recent evidence shows that distinct caspase pathways are activated during ischemia. We have identified the caspase-9/-6 pathway as responsible for neuronal dysfunction and death after ischemia. Our data show that targeting caspase-9 activity provides substantial neuroprotection following an ischemic insult. Moreover, we find that caspase-9 activity is required for two aspects of ischemic pathogenesis: 1) neuronal degeneration and 2) the development of cerebral edema. Edema is caused by a loss of vascular integrity, rather than death of endothelial cells and pericytes in the blood vessels (BV). The elimination of tight junctions between these cells allows extravasation of fluid from small intracranial BVs. Edema formation is a major contributor to death and disability in severe stroke. Medical therapies and surgical decompressive procedures have only minimally altered the natural history of this pathogenic process. In our studies, a cell permeant caspase-9 inhibitor, Pen1-XBIR3, reduces caspase-9 activity and concomitantly abolishes edema. This finding opens the question of whether caspase-9 activity is a direct cause of edema through the impairment of vascular integrity of small cerebral BVs. Our preliminary data suggest that active caspase-9 regulates edema by decreasing the expression of matrix metalloproteinase 9 (MMP-9). Our data also show that expression of the precursor of mature NGF, proNGF, increases during stroke. ProNGF is a high affinity ligand for p75NTR, and we have shown that signaling through p75NTR activates caspase-9. p75NTR is found in small BVs in the brain, and expression of p75NTR increases during stroke. Our preliminary data also show that activated caspase-9 is present in BVs, and that caspase-9 inhibition prevents the stroke-induced expression of MMP-9. We now propose the hypothesis that the development of edema in stroke is mediated by proneurotrophin (proNT) signaling through p75NTR, which activates caspase-9 in small BVs to cleave substrates vital to the integrity of the vessels. We will utilize in vivo an in vitro models to examine this hypothesis with the following Specific Aims: Aim 1: To determine if induction of proNTs triggers caspase-9 activation in small BVs. Aim 2: To determine if signaling via p75NTR activates caspase-9 and leads to edema. Aim 3: To determine how caspase-9 cleavage of substrates leads to loss of vascular integrity.

Our studies of cerebral ischemia have shown that caspase-9 is a critical mediator of edema, neuronal process loss and neuronal death. We have an RO1 funded project to study the mechanisms of edema in cerebral ischemia, NS081333 Mechanisms and Treatment of Cerebral Edema. In that grant we are examining how caspase-9 regulates cerebral edema due to cerebral ischemia. However, this project was written, reviewed and funded before we knew that there were caspase-9 conditional knockout mice available; Marc Tessier-Lavigne made the mice when he was at Genentech and kindly told us about these mice. We now propose to develop cell specific inducible caspase-9 knockout mice to examine the function of caspase-9 in the neurovascular unit during cerebral ischemia. The neurovascular unit, composed of neurons, glial cells and blood vessels, is a key target in cerebral ischemia. Our prior work suggests that while caspase-9 may function to orchestrate death in some cell types, neurons for example, there may be cell-specific functions of caspase-9 activity, such as a function in endothelial cells leading to edema but not to endothelial cell death. Caspase-9 null mice die in late embryogenesis with overgrowth of the frontal cortices. Thus these mice are not good tools to examine the functions of caspase-9 in adult animals or to study the cell specific functions of caspase-9. Genentech has developed a caspase-9 conditional null mouse to which we now have access. Our preliminary data show that tMCAo (transient middle cerebral artery occlusion) in rat and mouse models activates caspase-9 in neurons and in blood vessels. Moreover, activation in neurons leads to process loss and neuronal death while activation in blood vessels does not activate effector caspases or induce death. This raises the question of how activation of caspase-9 in each of these cell types leads to the development of stroke pathology. To approach this question, we propose to develop cell specific inducible knockouts of caspase-9 in neurons and endothelial cells. After obtaining the MTA for the caspase-9 floxed mice, we investigated the availability of inducible cell specific Cre mice. We are choosing to use inducible Cre because we are studying a model of adult disease and want to avoid any developmental phenotype that might occur with constitutive cell specific knockout of caspase-9. To target endothelial cells, we were able to get an MTA for Cdh5(PAC)-CreERT2 mice from Raif Adams. For neuronal knockout, Synapsin-driven CreERT2 mice are available from Jackson Labs. Due to the sequester, our RO1 project received a 12.5% cut for the entire project, thus there is not funding in the RO1 to develop these new tools that we are now proposing. The current RO3 grant proposal will allow us to generate these mice that will then be used in our funded RO1 project. These cell specific inducible knockout mice will be a valuable tool for our work and in the future will also be useful for other studies of caspase-9 function and caspase-9 cell specific substrates. Identifying the cell specific substrates of caspase-9 will provide additional disease targets for therapeutic interventions.

Summary Support is requested for training of 5 predoctoral trainee positions per year, in a university-wide visual science training program at the systems, cellular, and molecular level. Training by thirty-six faculty mentors focuses on analysis of visual processing, and cellular, molecular and genetic aspects of the normal and diseased eye, in both basic science and disease-oriented research. Twenty-five faculty work in Systems and Computation on the visual and oculomotor systems in humans, non-human primates, and other model systems, using neurophysiology, psychophysics, behavioral analysis, brain imaging, and computational modeling (Section 1). Six faculty in Section 2 (Development and Plasticity) focus on retinal cell specification and axon guidance, eye development, the blood-retina barrier, and biophysics and plasticity of cortical dendrites and circuitry. Eight faculty have research programs that touch on Molecular/genetic Approaches to the Normal and Diseased Eye (Section 3) with primary interests in retinal degeneration, retinoid processing, and the genetics, diagnostics and therapy of retinal disorders, with a focus on age-related macular degeneration and retinal edema. Although mentors are in different departments (Biological Sciences, Psychology, Statistics, Biomedical Engineering and Radiology, Ecology, Evolution and Environmental Biology on the Morningside campus; Neuroscience on the Manhattanville campus; and Genetics, Ophthalmology, Pathology & Cell Biology, Neurology, Medicine and Pediatrics on the medical school campus, multiple collaborations on interdisciplinary themes routinely support training and advances on vision The research carried out by the mentors and trainees matches the goals in NEI?s strategic plan for eye and vision research, including the Audacious Goals Initiative and 2020 Vision for the Future. Suppport is sought for up to three years for predoctoral students who have chosen their lab and mentor in vision studies. Trainees working on thesis projects related to the vision sciences are selected from selective graduate programs such as the Doctoral Program in Neurobiology and Behavior, and Integrated Program in Cellular, Molecular and Biomedical Sciences, both of which host MD-PhD students. Through activities such as didactic courses, training in rigor and reproducabilty, thesis committees, symposia, seminars, and the Greater New York Vision Club (VisioNYC), it is expected that faculty and trainees will interact, collaborate, and produce a new generation of vision scientists who will elucidate information processing, development, and diseases of the visual system.